C-Ski Efficiently Promotes Tissue Restoration After Traumatic Brain Injury

Background:Traumatic brain injury(TBI)is characterized by a high rate of incidence and mortality.Most survival patients suffer from disability and various neurological and psychiatric sequelae.The serious consequences of TBI are largely due to the difficulty of self-restoration of damaged brain tissue,which is a key and difficult point to TBI treatment.Generally speaking,in tissue trauma such as epidermal tissue injury,reparative cells such as fibroblasts will proliferate and fill the defective wound to achieve complete healing.However,in TBI,the proliferating cells were mainly supporting cells such as astrocytes.Although neurons in the injured area suffer a lot of losses,the remaining neurons are difficult to directly proliferate to make up for the loss.The reparative newborn mature neurons usually come from the migration of the newborn immature neurons in the non-injured area,but the number of regeneration is very limited and far from enough to compensate for the loss of neurons.In addition,hyperplastic astrocytes secret extracellular matrix,which is known as astrogliosis,constitute glial scars that hinder the migration of newborn neurons and the regeneration of axons.In addition,activated microglia are the most crucial cells mediating inflammation after TBI,which secrete a variety of inflammatory factors to participate in neuroinflammation.Deficiencies in newborn neuron generation,excessive astrogliosis and neuroinflammation are considered to be the important obstacle in tissue restoration after TBI.How to intervene in these factors to effectively promote tissue restoration after TBI has important clinical significance for the treatment and prognosis of TBI.c-Ski is an intracellular homologue of avian virus oncogene v-Ski,which distributes in various organs and tissues of mammalian animals.It participates in many pathophysiological processes in vivo,including the proliferation of fibroblasts,the regeneration of skeletal muscle and hepatic tissue,and the progression of many types of tumors.In the central nervous system,Our previous studies have found that c-Ski is highly expressed in injured dermal tissues and is involved in wound healing and scar formation.It is also associated with early wound inflammatory cell infiltration,the secretion of inflammatory factors such as interleukin-1beta(IL-1?)and tumor necrosis factor-alpha(TNF-?).However,it is not clear whether it is expressed in damaged brain tissue in TBI and plays a similar role.It has been reported that in the central nervous system,c-Ski participates in the proliferation of neurons during central nervous system development.Knockout ski gene can cause neurodevelopmental deficits in embryonic rats and decrease the number of neurons proliferating during development.In human 1p36 deletion syndrome,ski gene deletion is also manifested as delayed brain development and mental retardation.In addition,astrogliosis and glial scar formation are also fibrotic reactions,while c-Ski can inhibit many fibrotic reactions and scar formation.Therefore,we speculate that c-Ski may promote newborn neurons generation,inhibit astrogliosis and microglia activation,thereby promoting tissue restoration and functional recovery after TBI.Method:We explored the roles of c-Ski in the perilesion brain tissues after TBI using a moderate TBI mouse model:1.A 28-day observation of neuropathological change,MRI-based 3D-reconstructed cavity volume,c-Ski protein level and cell distribution types after TBI were analyzed to preliminarily speculate the roles of c-Ski.2.A c-Ski overexpression moderate TBI mice model was constructed by adenovirus transfection.Mice were divided into c-Ski adenovirus treating group and control adenovirus treating group.A 28-day observation of neuropathological change,MRI-based 3Dreconstructed cavity volume and neurobehavioral changes after TBI were analyzed to identify the roles of c-Ski in brain tissue restoration and functional recovery after TBI.3.In c-Ski adenovirus treating group and control adenovirus treating group,immunofluorescence,western blot and ELISA were used to observe the changes of newborn neurons,astrocytes and extracellular matrix,and microglia activation in perilesion tissue after TBI to determine whether c-Ski promotes tissue restoration by regulating the generation of newborn neurons,astrogliosis and microglia activation.Finally,mice were divided into c-Ski si RNA treating group and control si RNA treating group,similar evaluation was performed after c-Ski gene silencing using si RNA interference in TBI mice to reverse-verify the role of c-Ski in tissue restoration after TBI.Results:Chapter 1:1.The hemorrhage,edema and inflammation in perilesion regions began at 1 day and subsided at 3 days after TBI.The volume of defective lesion was not obvious at 3 days but expanded significantly at 7 days,and a huge cavity was formed at 28 days after TBI.2.There was a little c-Ski expression in normal brain tissues.After TBI,the level of c-Ski increased and peaked at 3 days,and then decreased.Increased c-Ski expression was found in mature neurons,newborn immature neurons,astrocytes and microglia.3.The defective lesion volume was relatively small at 3 days after injury,which is consistent with the peak time of c-Ski protein level.The defective lesion volume continued to expand from 7 days after injury,and at that time the c-Ski level began to decrease.The results suggest that the elevated c-Ski level may be involved in tissue restoration,and its effect may be related to its role on neurons,astrocytes and microglia.Chapter 2:1.A c-Ski overexpression moderate TBI mice model was constructed by a single injection of adenovirus in the perilesion regions immediately after injury.The c-Ski level peaked at 7 days after injury and continued until 28 days.Overexpress ed c-Ski was found in mature neurons,newborn immature neurons,astrocytes and microglia.2.At the pathological level,H&E staining showed that overexpressed c-Ski reduced the size of defective lesion at 3,7,14 and 28 days after injury.At the imaging level,3D Slicer based MRI imaging reconstruction showed that overexpressed c-Ski reduced the volume of defective lesion at 7,14 and 28 days after injury.At the behavioral level,overexpressed c-Ski alleviated the Neurological severity score at 3,7 and 14 days after injury.in open field experiment,overexpressed c-Ski facilitated the locomotive ability of mice at 7,14 and 28 days after injury.The anxiety degree at 28 days after injury was reduced in both open field experiment and elevated plus maze.These results clarify the role of c-Ski in promoting the restoration of damaged brain tissue after TBI.Chapter 3:1.Overexpressed c-Ski increased the remaining mature neurons in the perilesion regions at 3 and 28 days after TBI,Edu positive proliferated newborn immature neurons at 7 days after TBI,Edu positive proliferated mature neurons at 14 days after TBI,and the content of DCX protein in the perilesion regions at 3 and 7 days after TBI,indicating overexpressed c-Ski promotes the generation of newborn neurons.2.Overexpressed c-Ski reduced the astrocytes and Edu-positive proliferated astrocytes at 7 days after TBI,the content of GFAP and Laminin protein in the perilesion regions at 7 and 14 days after TBI,The distribution of CSPGS in the perilesion regions at 7 days after TBI,indicating that overexpressed c-Ski inhibits the astrogliosis and the secretion of extracellular matrix.3.Overexpressed of c-Ski reduced the activated microglia and the contents of IL-1? and TNF-? at 1 and 3 days after TBI,suggesting that overexpression of c-Ski inhibits the activation of microglia.4.After silencing Ski gene,the defective lesion volume increased at 3 and 7 days after TBI,the edu positive proliferated immature neurons decreased and proliferated astrocytes increased 3 days after TBI,indicating that reducing c-Ski level is harmful for tissue restoration after TBI.Conclusions:In summary,our study identified c-Ski as a new brain tissue restoration-related protein for the first time.Overexpressed c-Ski protein can improve damaged brain tissue restoration,facilitate locomotive function and reducing anxiety degree.These effects are related to the role of c-Ski in facilitating generation of newborn neurons to increase the neurons in perilesion regions,reducing astrogliosis and microglia activation.These results provide a promising strategy targeting c-Ski to promote structural restoration for the treatment of TBI,stroke,and various central nervous system injuries.